Devices and methods for separating magnetically labeled moieties in a sample

ABSTRACT

Devices for separating magnetically labeled moieties in a sample are provided. Aspects of the devices include a magnetic field source, a first magnetic field guide having a wedge-shaped portion with an apex edge, and a second magnetic field guide having a wedge-shaped portion with an apex edge. The apex edge of the first magnetic field guide is aligned substantially across from and parallel to the apex edge of the second magnetic field guide, and the device is configured to separate magnetically labeled moieties from non-magnetically labeled moieties in the sample. Also provided are methods of using the devices, as well as systems and kits configured for use with the devices and methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119 (e) this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 61/479,778filed Apr. 27, 2011, the disclosure of which application is hereinincorporated by reference.

INTRODUCTION

Magnetic immunoassays have been described in which analyte-specificantibodies conjugated to magnetic particles are used to magneticallylabel a target analyte to facilitate magnetic separation of the analytefrom the sample solution. Typically, after the magnetically labeledanalyte has been concentrated against the side or bottom of the samplechamber, the sample fluid is removed. Such assays require samplehandling fluidics to separate the captured analyte from the sample fluidand are inherently multi-step.

U.S. Pat. No. 5,945,281 describes a magnetic immunoassay in which alabeled target analyte is magnetically separated from a sample fluid andmoved from a sample chamber into a detection region for opticalanalysis. The sample is added to the sample chamber containing magneticcapture reagents and a label, such that the target analyte in the sampleforms a complex with the magnetic capture agent and the label. Anelectrical potential is applied to the complex to transport the complexto a detection region, and the presence of the complex in the detectionregion is determined.

U.S. Pat. Nos. 6,858,440; 6,645,777; 6,630,355; and 6,254,830, eachincorporated herein by reference, describe a magnetic focusingimmunosensor for magnetically concentrating pathogenic bacteria in afood sample onto the side of a sample container and optically detectingthe concentrated cells through the side of the sample container. Themagnetic focusing immunosensor includes a focusing magnet and fiberoptics attached to the side of the magnet for transmitting excitationand detection light.

SUMMARY

Devices and methods for separating magnetically labeled moieties in asample are provided. Embodiments of the device include magnetic fieldguides disposed on one or more magnetic field sources, where themagnetic field guides each have a wedge-shaped portion with an apexedge. The apex edges of the magnetic field guides are alignedsubstantially across from and parallel to each other. In certain cases,the device includes a conduit for carrying a sample flow in closeproximity to the apex edges of the magnetic field guides, such that thesample flow is substantially parallel to the apex edges of the magneticfield guides.

Embodiments of the present disclosure may achieve high efficiency, highflow rate and low cost magnetic separation of magnetically labeledmoieties in a sample. For example, embodiments of the present disclosuremay be used to separate cells and other molecules that are labeled withmagnetic particles from a biological fluid sample. In some instances,the efficiency of separation of the magnetically labeled moieties fromthe sample depends on the magnetic field and the magnetic field gradientproduced by the magnetic field source. In some cases, the force on themagnetic labels, and thus the efficiency of magnetic separation, dependson the product of the magnetic field and the magnetic field gradient.Thus, embodiments of the present disclosure may achieve both a highmagnetic field and high magnetic field gradient in the same spatiallocation, e.g., in the area between and/or proximal to the apex edges ofthe magnetic field guides through which the sample flows. The highmagnetic field and high magnetic field gradient produced by the devicemay increase the separation efficiency of the device, and thus allow forincreased sample flow rates through the device for high throughputseparation.

In certain embodiments, the device is configured to separatemagnetically labeled moieties from non-magnetically labeled moieties ina sample that flows through the device. In some cases, the deviceincludes a magnetic field source, such as a single magnetic fieldsource. In other cases, the device includes a first magnetic fieldsource and a second magnetic field source, which may be arranged onopposite sides of a fluid conduit. In some instances, the magnetic fieldsource may be a permanent magnet, rather than other types of magnetssuch as an electromagnet. Embodiments of the device that include apermanent magnet as the magnetic field source provide a sustainedmagnetic field without the need for an external power source, and thusmay be less complex and lower in cost to manufacture and operate than adevice that includes other types of magnets, such as electromagnets.

The device also includes a first magnetic field guide and a secondmagnetic field guide. In embodiments of the device that include onemagnetic field source, the first and second magnetic field guides may bedisposed on opposite sides of the magnetic field source. In otherembodiments of the device that include first and second magnetic fieldsources, the first magnetic field guide may be disposed on a surface ofthe first magnetic field source facing the second magnetic field source,and the second magnetic field guide that may be disposed on a surface ofthe second magnetic field source facing the first magnetic field source.The first magnetic field guide and the second magnetic field guide eachhave a wedge-shaped portion with an apex edge. The first and secondmagnetic field guides are arranged such that the apex edge of the firstmagnetic field guide is aligned substantially across from and parallelto the apex edge of the second magnetic field guide. In some instances,the magnetic field guides are soft magnets.

Each magnetic field guide has a wedge-shaped portion and may beconfigured to direct the magnetic flux from the associated magneticfield source towards the area proximal to the apex edge of the magneticfield guide. In some cases, the wedge-shaped portion of the magneticfield guide focuses the magnetic flux from the interface between themagnetic field source and the magnetic field guide, where the interfacehas a relatively large cross-sectional area, to the apex edge of themagnetic field guide, which has a relatively small cross-sectional area.The wedge-shaped portion of the magnetic field guide may be configuredto focus the magnetic flux from the associated magnetic field sourcewith minimal magnetic flux leakage during the transmission of themagnetic flux through the magnetic field guide. In certain embodiments,the tapered wedge shape portion of the magnetic field guide focuses themagnetic flux from the associated magnetic field source, resulting in anincrease in the magnetic flux from the magnetic field source in the areaproximal to (e.g., near and/or between) the apex edge of the magneticfield guide. The resulting high magnetic field strength and highmagnetic field gradient in the area proximal to (e.g., near and/orbetween) the apex edge of the magnetic field guide may increase theefficiency of the separation of magnetically labeled moieties fromnon-labeled moieties in the sample being analyzed.

A fluid conduit for directing the flow of a sample fluid through thedevice may be positioned in the area between or proximal to the apexedges of the magnetic field guides such that a longitudinal axis of theconduit is substantially parallel to the apex edges of the first andsecond magnetic field guides. As such, in certain embodiments, thesample fluid is directed by the conduit to flow in close proximity toand substantially parallel to the apex edges of the magnetic fieldguides. Positioning the conduit in close proximity to and substantiallyparallel to the apex edges of the magnetic field guides may maximize theamount of time the sample fluid flow is exposed to the locally highmagnetic field and magnetic field gradient in the area proximal to theapex edges of the magnetic field guides, and thus may increase theseparation efficiency of the device.

As the sample flows through the conduit, magnetically labeled moietiesin the sample are retained in the conduit by the magnetic field producedby the device. Non-labeled moieties in the sample are not retained inthe conduit and flow through the device. The retained magneticallylabeled moieties can be recovered by positioning the conduit away fromthe magnetic field and flushing the magnetically labeled moieties fromthe conduit. The conduit may be positioned in the magnetic field andpositioned away from the magnetic field either manually orautomatically. In some cases, the conduit may be disposable, such as asingle-use conduit, which may be suitable for clinical applications.

Aspects of the present disclosure further include systems for separatingmagnetically labeled moieties in a sample, where the system includes oneor more magnetic separation devices as described herein. In certainembodiments, the system includes two magnetic separation devices, suchas a first magnetic separation device and a second magnetic separationdevice arranged downstream from the first magnetic separation device.The apex edges of the first and second magnetic field guides of thefirst magnetic separation device may have substantially the sameprofiles as the apex edges of the first and second magnetic field guidesof the second magnetic separation device. For example, the apex edges ofthe first and second magnetic field guides of the first magneticseparation device and the apex edges of the first and second magneticfield guides of the second magnetic separation device may each have alinear profile. In other embodiments, the apex edges of the first andsecond magnetic field guides of the first magnetic separation devicehave different profiles from the apex edges of the first and secondmagnetic field guides of the second magnetic separation device. Forinstance, the apex edges of the first and second magnetic field guidesof the first magnetic separation device may each have a linear profileand the apex edges of the first and second magnetic field guides of thesecond magnetic separation device may each have a saw-tooth profile.

Accordingly, embodiments of the present disclosure include a device forseparating magnetically labeled moieties in a sample. The deviceincludes a magnetic field source, a first magnetic field guide having awedge-shaped portion with an apex edge, and a second magnetic fieldguide having a wedge-shaped portion with an apex edge. One or more ofthe first and second magnetic field guides is configured to increase amagnetic flux from the magnetic field source, the apex edge of the firstmagnetic field guide is aligned substantially across from and parallelto the apex edge of the second magnetic field guide, and the device isconfigured to separate magnetically labeled moieties fromnon-magnetically labeled moieties in the sample.

Embodiments of the device may also include that both the first andsecond magnetic field guides are configured to increase the magneticflux from the magnetic field source.

Embodiments of the device may also include that a cross-sectionalprofile of one of more of the first magnetic field guide and the secondmagnetic field guide tapers to a point at the apex edge.

Embodiments of the device may also include that one of more of the firstmagnetic field guide and the second magnetic field guide has a roundedcross-sectional profile at the apex edge.

Embodiments of the device may also include that the apex edge of thefirst magnetic field guide is a substantially uniform distance along itslength from the apex edge of the second magnetic field guide.

Embodiments of the device may also include that the apex edge of thefirst magnetic field guide is a distance from the apex edge of thesecond magnetic field guide ranging from 0.1 mm to 5 mm.

Embodiments of the device may also include that the apex edge of thefirst magnetic field guide and the apex edge of the second magneticfield guide each have a linear profile.

Embodiments of the device may also include that the apex edge of thefirst magnetic field guide and the apex edge of the second magneticfield guide each have a saw-tooth profile.

Embodiments of the device may also include that the first magnetic fieldguide and the second magnetic field guide each have an apex angle of 90degrees or less.

Embodiments of the device may also include that the first magnetic fieldguide and the second magnetic field guide each include a soft magnet.

Embodiments of the device may also include a conduit positioned betweenthe first magnetic field guide and the second magnetic field guide andconfigured to direct a flow of the sample through the device.

Embodiments of the device may also include that the conduit ispositioned such that a longitudinal axis of the conduit is substantiallyparallel to a longitudinal axis of the first magnetic field guide and alongitudinal axis of the second magnetic field guide.

Embodiments of the device may also include that the first magnetic fieldguide and the second magnetic field guide are disposed on opposite sidesof the magnetic field source.

Embodiments of the device may also include a second magnetic fieldsource.

Embodiments of the device may also include that the first magnetic fieldguide is disposed on a surface of the magnetic field source facing thesecond magnetic field guide and is configured to increase the magneticflux from the magnetic field source, and the second magnetic field guideis disposed on a surface of the second magnetic field source facing thefirst magnetic field guide and is configured to increase a magnetic fluxfrom the second magnetic field source.

Embodiments of the device may also include that the device is configuredto automatically position the conduit in the device.

Embodiments of the device may also include that the conduit has atapered cross-sectional shape such that the cross-sectional dimension ofthe conduit proximal to the apex edges of the first and second magneticfield guides is less than the cross-sectional dimension distal to theapex edges of the first and second magnetic field guides.

Embodiments of the device may also include that the conduit issubstantially free from magnetic gradient enhancing materials.

Embodiments of the device may also include that the conduit isconfigured to be positionable away from the magnetic field.

Embodiments of the device may also include that the magnetic fieldsource includes a permanent magnet.

Embodiments of the device may also include that the permanent magnetincludes a rare-earth magnet.

In some embodiments, a method of separating magnetically labeledmoieties in a sample is provided. The method includes positioning in amagnetic separation device a conduit configured to direct a flow of asample through the magnetic separation device, and applying a magneticfield to separate magnetically labeled moieties from non-magneticallylabeled moieties in the sample. The magnetic separation device includesa magnetic field source, a first magnetic field guide having awedge-shaped portion with an apex edge, and a second magnetic fieldguide having a wedge-shaped portion with an apex edge, where one or moreof the first and second magnetic field guides is configured to increasea magnetic flux from the magnetic field source, and the apex edge of thefirst magnetic field guide is proximal to and substantially parallel tothe apex edge of the second magnetic field guide.

Embodiments of the method may also include that the positioning includespositioning the conduit in the device such that a longitudinal axis ofthe conduit is substantially parallel to a longitudinal axis of thefirst magnetic field guide and a longitudinal axis of the secondmagnetic field guide.

Embodiments of the method may also include positioning the conduit awayfrom the magnetic field, and recovering the magnetically labeledmoieties retained in the conduit.

Embodiments of the method may also include that the positioning theconduit away from the magnetic field includes removing the conduit fromthe device.

Embodiments of the method may also include that the positioning theconduit away from the magnetic field includes moving the magnetic fieldsource away from the conduit.

Embodiments of the method may also include that the recovering includesflushing the magnetically labeled moieties from the conduit.

Embodiments of the method may also include specifically attaching amagnetic label to target moieties in the sample prior to applying themagnetic field to the sample.

Embodiments of the method may also include that the sample includes abiological sample.

In some embodiments, a system for separating magnetically labeledmoieties in a sample is provided. The system includes one or moremagnetic separation devices for separating magnetically labeled moietiesin the sample, where each of the one or more magnetic separation devicesincludes a magnetic field source, a first magnetic field guide having awedge-shaped portion with an apex edge, and a second magnetic fieldguide having a wedge-shaped portion with an apex edge. One or more ofthe first and second magnetic field guides is configured to increase amagnetic flux from the magnetic field source, the apex edge of the firstmagnetic field guide is proximal to and substantially parallel to theapex edge of the second magnetic field guide, and the device isconfigured to separate magnetically labeled moieties fromnon-magnetically labeled moieties in the sample. The system alsoincludes a conduit positioned in the magnetic separation device andconfigured to direct a flow of the sample through the magneticseparation device.

Embodiments of the system may also include that the conduit ispositioned such that a longitudinal axis of the conduit is substantiallyparallel to a longitudinal axis of the first magnetic field guide and alongitudinal axis of the second magnetic field guide.

Embodiments of the system may also include that the system includes onemagnetic separation device.

Embodiments of the system may also include that the system includes afirst magnetic separation device and a second magnetic separation devicearranged downstream from the first magnetic separation device.

Embodiments of the system may also include that the apex edges of thefirst and second magnetic field guides of the first magnetic separationdevice have substantially the same profiles as the apex edges of thefirst and second magnetic field guides of the second magnetic separationdevice.

Embodiments of the system may also include that the apex edges of thefirst and second magnetic field guides of the first magnetic separationdevice and the apex edges of the first and second magnetic field guidesof the second magnetic separation device each have a linear profile.

Embodiments of the system may also include that the apex edges of thefirst and second magnetic field guides of the first magnetic separationdevice have different profiles from the apex edges of the first andsecond magnetic field guides of the second magnetic separation device.

Embodiments of the system may also include that the apex edges of thefirst and second magnetic field guides of the first magnetic separationdevice each have a linear profile and the apex edges of the first andsecond magnetic field guides of the second magnetic separation deviceeach have a saw-tooth profile.

Embodiments of the system may also include a flow cytometer arrangeddownstream from the one or more magnetic separation devices.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(a) shows a schematic of a front view of a magnetic separationdevice that includes two magnetic field sources, according toembodiments of the present disclosure. FIG. 1(b) shows a schematic of aside view of a magnetic separation device that includes two magneticfield sources, according to embodiments of the present disclosure. FIG.1(c) shows a schematic of a three-dimensional perspective view of amagnetic separation device that includes two magnetic field sources,according to embodiments of the present disclosure.

FIG. 2(a) shows a schematic of a longitudinal cross section of aconduit, according to embodiments of the present disclosure. FIG. 2(b)shows a schematic of a front view of a conduit, according to embodimentsof the present disclosure.

FIGS. 3(a) and 3(b) show schematics of front views of a conduitpositioned between the magnetic field guides in a magnetic separationdevice, according to embodiments of the present disclosure.

FIG. 4(a) shows a schematic of a front view of a magnetic separationdevice, according to embodiments of the present disclosure. FIG. 4(b)shows a schematic of a side view of a magnetic separation device havingmagnetic field guides with a saw-tooth shaped profile, according toembodiments of the present disclosure.

FIG. 5 shows a schematic of a conduit positioned transverse to themagnetic field guides, according to embodiments of the presentdisclosure.

FIGS. 6(a), 6(b) and 6(c) show graphs of a simulated magnetic field(FIG. 6(a)), magnetic field gradient (FIG. 6(b)), and product of themagnetic field and absolute magnetic field gradient (FIG. 6(c)) acrossthe gap between the magnetic field guides as shown in FIG. 1(a) for amagnetic separation device with a distance between the apex edges of themagnetic field guides of 1.4 mm, according to embodiments of the presentdisclosure. The x-axis is along the center of the gap from left to rightbetween the apex edges of the magnetic field guides, as shown in FIG.3(a).

FIG. 7 shows a schematic of a system including a magnetic separationdevice, an acoustic concentrator and a flow cytometer, according toembodiments of the present disclosure.

FIG. 8(a) shows a schematic of a front view of a magnetic separationdevice that includes one magnetic field source, according to embodimentsof the present disclosure. FIG. 8(b) shows a schematic of athree-dimensional perspective partial view of a magnetic separationdevice that includes one magnetic field source, according to embodimentsof the present disclosure.

FIG. 9 shows a schematic cross-section of a conduit operatively coupledto a conduit holder and positioned in close proximity to the apex edgesof the magnetic field guides in a magnetic separation device, accordingto embodiments of the present disclosure.

FIG. 10(a) shows a schematic of a front view of a conduit operativelycoupled to a conduit holder, according to embodiments of the presentdisclosure. FIG. 10(b) shows a schematic of a three-dimensionalperspective view of a conduit operatively coupled to a conduit holder,according to embodiments of the present disclosure.

FIG. 11 shows a three-dimensional perspective view of a conduitoperatively coupled to a conduit holder and positioned in a magneticseparation device, according to embodiments of the present disclosure.

DETAILED DESCRIPTION

Devices for separating magnetically labeled moieties in a sample areprovided. Aspects of the devices include a magnetic field source, afirst magnetic field guide having a wedge-shaped portion with an apexedge, and a second magnetic field guide having a wedge-shaped portionwith an apex edge. The apex edge of the first magnetic field guide isaligned substantially across from and parallel to the apex edge of thesecond magnetic field guide, and the device is configured to separatemagnetically labeled moieties from non-magnetically labeled moieties inthe sample. Also provided are methods of using the devices, as well assystems and kits configured for use with the devices and methods.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating un-recited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodiments arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace operableprocesses and/or devices/systems/kits. In addition, all sub-combinationslisted in the embodiments describing such variables are alsospecifically embraced by the present invention and are disclosed hereinjust as if each and every such sub-combination of chemical groups wasindividually and explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

In further describing embodiments of the present disclosure, aspects ofembodiments of the devices will be described first in greater detail.Next, embodiments of methods, systems and kits that may be used with thedevices are reviewed.

Devices

Provided are devices for separating magnetically labeled moieties in asample. The device may be configured to separate magnetically labeledmoieties from non-magnetically labeled moieties (e.g., moieties that arenot associated with a magnetic label) in the sample. In certaininstances, the device separates magnetically labeled moieties ofinterest from moieties that are not of interest (e.g., moieties that arenot magnetically labeled) by retaining the magnetically labeled moietiesin the device while not retaining moieties that are not of interest.Because the moieties of interest are magnetically labeled, the devicemay be configured to retain the magnetically labeled moieties in thedevice by attracting the magnetically labeled moieties to a magneticfield source in the device and retaining the magnetically labeledmoieties in the device. In other cases, the device separatesmagnetically labeled moieties that are not of interest from moietiesthat are of interest (e.g., moieties of interest that are notmagnetically labeled) by retaining the magnetically labeled moietiesthat are not of interest in the device while not retaining moieties thatare of interest. In these embodiments, because the moieties of interestare not magnetically labeled, the moieties of interest are not retainedin the device and flow through the device. The device may be configuredto retain the magnetically labeled moieties that are not of interest inthe device by attracting the magnetically labeled moieties to a magneticfield source in the device and retaining the magnetically labeledmoieties that are not of interest in the device.

The device may be configured as a flow-through device for analyzingliquid samples. By “flow-through” is meant that a liquid sample mayenter the device through an inlet, be carried through the device in aflow path, such as a conduit, and then exit the device through anoutlet. The device may be configured to carry a continuous stream of thesample through the device and continuously separate magnetically labeledmoieties in the sample as the sample flows through the device. Incertain embodiments, the device is configured to have a flow rate of 1μL/min or more, such as 10 μL/min or more, including 50 μL/min or more,or 100 μL/min or more, or 200 μL/min or more, or 300 μL/min or more, or400 μL/min or more, or 500 μL/min or more, or 750 μL/min or more, or 1mL/min or more, or 2 mL/min or more, or 5 mL/min or more, or 10 mL/minor more.

The magnetic separation device may be configured to separatemagnetically labeled moieties from a simple sample or complex sample. By“simple sample” is meant a sample that includes one or more magneticallylabeled moieties and few, if any, other molecular species apart from thesolvent. By “complex sample” is meant a sample that includes the one ormore magnetically labeled moieties of interest and also includes manyother molecules that are not of interest, such as different proteins,cells, and the like. In certain embodiments, the complex sample is ablood sample, by which is meant blood or a fraction thereof, e.g.,serum. In certain embodiments, the complex sample is a serum sample. Incertain embodiments, the complex sample assayed using the devicesdisclosed herein is one that includes 10 or more, such as 20 or more,including 100 or more, e.g., 10³ or more, 10⁴ or more (such as 15,000;20,000 or even 25,000 or more) distinct (i.e., different) molecularentities that differ from each other in terms of molecular structure.

In certain embodiments, the device is configured to separatemagnetically labeled moieties from a biological sample. A “biologicalsample” encompasses a variety of sample types obtained from an organismand can be used in a diagnostic or monitoring assay. For example, abiological sample encompasses blood, blood-derived samples, and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. A biological sample may also include samples that havebeen manipulated in any way after their procurement, such as bytreatment with reagents, solubilization, enrichment for certaincomponents, or labeling (e.g., labeling with a magnetic label). The term“biological sample” encompasses a clinical sample, and also includescells in culture, cell supernatants, cell lysates, serum, plasma,cerebrospinal fluid, urine, saliva, biological fluid, and tissuesamples.

Moieties of interest may include any moiety that can be stablyassociated with a magnetic label detectable by the devices disclosedherein. By “stably associated” is meant that the magnetic label and themoiety of interest maintain their position relative to each other inspace under the conditions of use, e.g., under the assay conditions. Assuch, the magnetic label and the moiety of interest can benon-covalently or covalently stably associated with each other. Examplesof non-covalent associations include non-specific adsorption, bindingbased on electrostatic (e.g. ion, ion pair interactions), hydrophobicinteractions, hydrogen bonding interactions, specific binding through aspecific binding pair member covalently attached to the moiety ofinterest or the magnetic label, combinations thereof, and the like.Examples of covalent binding include covalent bonds formed between themagnetic label and a functional group present on the moiety of interest,e.g. —OH, where the functional group may be naturally occurring orpresent as a member of an introduced linking group. Accordingly, themagnetic label may be adsorbed, physisorbed, chemisorbed, or covalentlyattached to the surface of the moiety of interest.

Magnetic Field Source

Aspects of embodiments of the device for separating magnetically labeledmoieties in a sample include one or more magnetic field sources. Themagnetic field source may be configured to produce a magnetic field. Incertain cases, the magnetic field source produces an inhomogeneousmagnetic field. By “inhomogeneous” is meant that the magnetic field hasa magnetic field gradient, where the strength of the magnetic field isdifferent depending on the position within the magnetic field. Forinstance, the magnetic field may have a magnetic field gradient, wherethe magnetic field strength is greater at one area and graduallydecreases at positions further away from that area. Thus, the magneticfield source may be configured to produce a magnetic field having amagnetic field gradient.

In some instances, the device is configured to produce a magnetic fieldsufficient to separate the magnetically labeled moieties in the sample.The ability of the magnetic field to separate the magnetically labeledmoieties in the sample may depend on various parameters, such as themagnetic field strength, the magnetic field gradient, the type ofmagnetic label, the size of the magnetic label, the distance between themagnetically labeled moieties and the magnetic field source, etc. Incertain instances, the force the magnetic field is able to exert on amagnetic label is proportional to the magnetic field strength and themagnetic field gradient. In some cases, the magnetic field source isconfigured to produce a magnetic field having a magnetic forcesufficient to separate magnetically labeled moieties formnon-magnetically labeled moieties in the sample. For example, themagnetic field source may be configured to produce a magnetic fieldhaving a magnetic field gradient such that the product of the magneticfield and the magnetic field gradient is sufficient to separatemagnetically labeled moieties from non-magnetically labeled moieties inthe sample.

The magnetic field source may be of any shape that may facilitate theseparation of the magnetically labeled moieties from thenon-magnetically labeled moieties in the sample. For example, themagnetic field source may be elongated, such that the magnetic fieldsource has a length that is greater than the transverse width of themagnetic field source.

In certain embodiments, the device may be configured to direct a flow ofthe sample through the device such that the sample flow is proximal tothe magnetic field source. Minimizing the distance between the magneticfield source and the sample, and thereby minimizing the distance betweenthe magnetic field source and the magnetically labeled moieties in thesample may facilitate the retention of the magnetically labeled moietiesin the device. In some cases, the device is configured to direct theflow of the sample through the device to maximize the length of the flowpath that is proximal to the magnetic field source. For example, thedevice may be configured to direct the flow of the sample through thedevice such that the sample flow is substantially parallel to thelongitudinal axis of the magnetic field source.

In certain embodiments, the device includes one magnetic field source.In some cases, the magnetic field source is configured to produce amagnetic field sufficient to separate magnetically labeled moieties formnon-magnetically labeled moieties in the sample. For example, themagnetic field source may be configured to produce a magnetic fieldsufficient to retain the magnetically labeled moieties in the device. Inembodiments that include one magnetic field source, the device may beconfigured to direct the flow of the sample through the device such thatthe sample flows through an area near the magnetic field source. In somecases, the device is configured to direct the flow of the sample throughthe device such that the sample flow is substantially parallel to alongitudinal axis of the magnetic field source. The device may also beconfigured to direct the flow of the sample through an area near themagnetic field source, where the magnetic field and magnetic fieldgradient produced by the magnetic field source may be strongest.

In other embodiments, the device includes two magnetic field sources,although the device may include any number of magnetic field sources,such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 ormore magnetic field sources as desired. For instance, the device mayinclude a first magnetic field source and a second magnetic fieldsource. In some cases, the first magnetic field source and the secondmagnetic field source are configured to produce an inhomogeneousmagnetic field (e.g., a magnetic field having a magnetic field gradient)sufficient to separate magnetically labeled moieties formnon-magnetically labeled moieties in the sample. The first magneticfield source and the second magnetic field source may be configured toproduce a magnetic field sufficient to retain the magnetically labeledmoieties in the device. In certain embodiments, the first and secondmagnetic field sources are arranged such that a magnetic field isproduced in an area between the magnetic field sources. As such, thefirst and second magnetic field sources may be configured to produce amagnetic field sufficient to retain the magnetically labeled moieties inan area between the magnetic field sources.

In certain embodiments, the first magnetic field source has a surfacethat faces the second magnetic field source, and the second magneticfield source has a surface that faces the first magnetic field source,such that these two surfaces are opposing each other. The surface of thefirst magnetic field source that faces the second magnetic field sourcemay be substantially planar. Similarly, the surface of the secondmagnetic field source that faces the first magnetic field source may besubstantially planar. In some instances, the surfaces of the firstmagnetic field source and the second magnetic field source that faceeach other are substantially parallel to each other. In these instances,the opposing surfaces of the first and second magnetic field sources maybe a substantially uniform distance from each other. In otherembodiments, the opposing surfaces of the first and second magneticfield sources are not parallel to each other, such that one end of thefirst magnetic field source is closer to the second magnetic fieldsource than the opposite end of the first magnetic field source. In somecases, the first magnetic field source and the second magnetic fieldsource are both elongated. The longitudinal axis of the first magneticfield source may be substantially parallel to the longitudinal axis ofthe second magnetic field source.

In embodiments that include a first magnetic field source and a secondmagnetic field source, the magnetization vectors of the first magneticfield source and the second magnetic field source may be aligned insubstantially the same direction. In some instances, having a firstmagnetic field source and a second magnetic field source withmagnetization vectors aligned in substantially the same directionfacilitates the formation of a magnetic field and a magnetic fieldgradient in an area between the first and second magnetic field sources.In certain embodiments, the magnetization vector of the first magneticfield source is substantially perpendicular to the surface that facesthe second magnetic field source. In some cases, the magnetizationvector of the second magnetic field source is substantiallyperpendicular to the surface that faces the first magnetic field source.In certain instances, the magnetization vectors of the first and secondmagnetic field sources are both substantially perpendicular to thesurfaces of the first and second magnetic field sources that face eachother and are aligned in substantially the same direction.

In embodiments that include first and second magnetic field sources, thedevice may be configured to direct the flow of the sample through thedevice such that the sample flows through an area between the firstmagnetic field source and the second magnetic field source. In somecases, as described above, the first and the second magnetic fieldsources are aligned such that their longitudinal axes are substantiallyparallel. In these cases, the device may be configured to direct theflow of the sample through the device such that the sample flow issubstantially parallel to the longitudinal axes of the first and secondmagnetic field sources. The device may also be configured to direct theflow of the sample through an area between the first and second magneticfield sources, where the magnetic field and magnetic field gradientproduced by the first and second magnetic field sources may bestrongest.

The magnetic field source may include a permanent magnet, anelectromagnet, a superconducting magnet, combinations thereof, and thelike. In certain embodiments, the magnetic field source includes one ormore permanent magnets. A “permanent magnet” is a magnetic material thathas a persistent magnetic field such that the magnetic field does notsubstantially decrease over time. In contrast, the term “soft magnet”refers to a material that can be magnetized in the presence of anapplied external magnetic field, but whose magnetism substantiallydecreases when the external magnetic field is removed. In embodimentswhere the magnetic field source includes one or more permanent magnets,the use of permanent magnets may facilitate the production of a magneticfield without the need for external energy input into the device topower the magnetic field source. In some cases, a permanent magnet costsless than an electromagnet or a superconducting magnet that produces amagnetic field with a substantially similar magnetic field strength andmagnetic field gradient. In these cases, the use of a permanent magnetmay reduce the cost of the magnetic field source, and thus reduce theoverall cost of the device. In certain cases, when the magnetic fieldsource includes one or more permanent magnets, the use of a permanentmagnet may facilitate the production of a device that is less complexthan a device that includes an electromagnet and/or a superconductingmagnet. For example, embodiments of the device that include a permanentmagnet may not need to include components associated with anelectromagnet and/or a superconducting magnet, such as a power source,electrical circuits associated with the magnetic field source, coolingcomponents associated with the electromagnet and/or superconductingmagnet, temperature sensors, and the like.

In some instances, the magnetic field source includes two or morepermanent magnets. The permanent magnets may be of any desirable shape,and in some instances may be cube or bar-shaped permanent magnets. Incertain cases, the magnetic field source may have a length ranging from1 cm to 100 cm, such as from 1 cm to 75 cm, including from 1 cm to 50cm, or from 1 cm to 25 cm, or from 1 cm to 10 cm, or from 5 cm to 10 cm,for example from 5 cm to 6 cm; a width ranging from 0.1 cm to 100 cm,such as from 0.1 cm to 75 cm, including from 0.1 cm to 50 cm, or from0.1 cm to 25 cm, or form 0.1 cm to 10 cm, or from 0.1 cm to 5 cm, orfrom 0.1 cm to 2 cm, or from 0.5 cm to 2 cm, for example from 1 cm to1.5 cm; and a height ranging from 0.1 cm to 100 cm, such as from 0.1 cmto 75 cm, including from 0.1 cm to 50 cm, or from 0.1 cm to 25 cm, orfrom 0.1 cm to 10 cm, or from 0.1 cm to 5 cm, or from 0.1 cm to 2 cm, orfrom 0.5 cm to 2 cm, for example from 1 cm to 1.5 cm.

The magnetic field source may be a permanent magnet, such as arare-earth magnet. Rare-earth magnets include, but are not limited to,samarium-cobalt magnets (e.g., SmCo₅), neodymium alloy (NdFeB) magnets(e.g., Nd₂Fe₁₄B), and the like.

In certain embodiments, the magnetic field source produces a magneticfield ranging from 0.01 T to 10 T, or from 0.01 T to 5 T, or from 0.01 Tto 2 T, or from 0.1 T to 2 T, or from 0.1 T to 1.5 T, including from 0.1T to 1 T. In some cases, the magnetic field source is configured toproduce a magnetic field with a magnetic field gradient (e.g., anabsolute field gradient) ranging from 0.1 T/mm to 10 T/mm, such as from0.1 T/mm to 7 T/mm, or from 0.1 T/mm to 5 T/mm, or from 0.1 T/mm to 3T/mm, such as from 0.1 T/mm to 2 T/mm, including from 0.1 T/mm to 1T/mm. In certain instances, the magnetic field source produces amagnetic field having a magnetic field gradient such that the product ofthe magnetic field and the magnetic field gradient (e.g., absolute fieldgradient) ranges from 0.001 T²/mm to 100 T²/mm, such as from 0.01 T²/mmto 75 T²/mm, including from 0.1 T²/mm to 50 T²/mm, or from 0.1 T²/mm to25 T²/mm, or from 0.1 T²/mm to 10 T²/mm, or from 0.1 T²/mm to 5 T²/mm,or from 0.1 T²/mm to 3 T²/mm, such as from 0.1 T²/mm to 2 T²/mm,including from 0.1 T²/mm to 1 T²/mm.

Magnetic Field Guides

Aspects of the device for separating magnetically labeled moieties in asample also include one or more magnetic field guides. The magneticfield guide may be configured to direct the magnetic field from themagnetic field source to the sample flow path. In certain instances, themagnetic field guide is configured to focus the magnetic field producedby the magnetic field source. The magnetic field guide may focus themagnetic field by increasing the magnetic flux of the magnetic fieldsource, where the magnetic flux is the amount of magnetic field (e.g.,the magnetic field density) that passes through a given surface area.The magnetic flux may depend on the magnetic field strength, the area ofthe surface and the angle between the magnetic field and the surface.For example, the magnetic field guide may focus the magnetic field, andthus increase the magnetic flux, by directing the magnetic field througha smaller area. In some cases, directing the magnetic field through asmaller area increases the magnetic field density, thus resulting in anincrease in the magnetic flux. The magnetic field source and themagnetic field guide may be configured to produce a magnetic fluxsufficient to separate magnetically labeled moieties fromnon-magnetically labeled moieties in a sample. In some instances, themagnetic field guide is configured to produce a magnetic field having amagnetic flux density ranging from 0.01 T to 10 T, or from 0.01 T to 5T, or from 0.01 T to 2 T, such as from 0.1 T to 2 T, including from 0.5T to 1.5 T.

In certain cases, the magnetic field guide is configured to direct themagnetic field from the magnetic field source to the sample flow pathwith minimal loss in magnetic flux. In some cases, the magnetic fieldguide is configured to direct the magnetic field from the magnetic fieldsource to the sample flow path with substantially no loss in magneticflux. Without any intent to be bound by theory, the magnetic field guidemay be configured to minimize the decrease in magnetic flux due to theself-demagnetization fields present in a soft magnet near the surfacesof the soft magnet. For example, the magnetic field guide may beconfigured to direct the magnetic field from the magnetic field sourceto the sample flow path with a decrease in magnetic flux of 50% or lessfrom the initial magnetic flux, such as 40% or less, including 30% orless, or 25% or less, or 20% or less, or 15% or less, or 10% or less, or7% or less, or 5% or less, for example 3% or less, or 2% or less, or 1%or less from the initial magnetic flux.

In certain embodiments, the magnetic field guide is configured to focusthe magnetic field by having portion with a tapered shape and bydirecting the magnetic field from the magnetic field source through thetapered portion of the magnetic field guide. By “tapered” is meant thata portion of the magnetic field guide has a wider end with a largercross-sectional area and the cross-sectional area of the portion of themagnetic field guide becomes progressively smaller towards a narroweropposing end of the magnetic field guide. For example, the magneticfield guide may have a wedge-shaped portion, where the base of thewedge-shaped portion has an area. Cross-sections of the wedge-shapedportion taken parallel to the base of the wedge-shaped portion will haveprogressively smaller areas towards the end of the wedge-shaped portionopposite from the base (i.e., towards the apex edge of the wedge-shapedportion).

In some instances, the magnetic field guide has a wedge-shaped portionand is configured to direct the magnetic field from the base of thewedge-shaped portion to the apex edge of the wedge-shaped portion.Directing the magnetic field from the base of the wedge-shaped portionto the apex edge of the wedge-shaped portion may facilitate an increasein the magnetic flux of the magnetic field from the magnetic fieldsource, as described above. An increase in the magnetic flux at the apexedge of the wedge-shaped portion of the magnetic field guide may producea higher magnetic field and a higher magnetic field gradient proximal tothe apex edge of the magnetic field guide than would be present in theabsence of the magnetic field guide. Other tapered shapes for themagnetic field guide are possible, such as, but not limited to, pyramid,cone, frustum, combinations thereof, and the like.

In some instances, the magnetic field guide includes a portion thattapers to a point or an edge (e.g., the apex edge). For example, across-sectional profile of the magnetic field guide may taper to a pointat the apex edge of the magnetic field guide. In other embodiments, thecross-sectional profile of the magnetic field guide tapers to a roundededge such that the apex edge has a rounded (e.g., arcuate)cross-sectional profile at the apex edge. The term “wedge-shaped” asused herein is meant to include embodiments of the magnetic field guidethat have an apex edge with a cross-sectional profile that tapers to apoint at the apex edge. The term “wedge-shaped” also includesembodiments of the magnetic field guide that have an apex edge with across-sectional profile that does not taper to a point at the apex edge.For example, the apex edge of the magnetic field guide may have across-sectional profile that is rounded, truncated, blunted, and thelike. The apex edge of the magnetic field guide may have a width that isapproximately the same as the width (or diameter) of a conduitpositioned adjacent the apex edge of the magnetic field guide. Incertain embodiments, the apex edge of the magnetic field guide has awidth that is less than the width (or diameter) of the conduit. In somecases, the width of the apex edge of the magnetic field guide is 5 mm orless, such as 4 mm or less, or 3 mm or less, or 2 mm or less, or 1 mm orless, or 0.5 mm or less, or 0.1 mm or less.

In certain instances, the apex edge of a wedge-shaped portion of themagnetic field guide has an apex angle, where the apex angle is theangle between the two faces of magnetic field guide that meet at theapex edge. In some cases, the apex angle is 150 degrees or less, or 135degrees or less, such as 120 degrees or less, or 105 degrees or less,including 90 degrees or less, or 75 degrees or less, or 60 degrees orless, or 45 degrees or less, for example 30 degrees or less. In someembodiments, the apex angle is 60 degrees.

In certain embodiments, the apex edge of the magnetic field guide may besubstantially parallel to a longitudinal axis of the magnetic fieldguide. In addition, the apex edge of the magnetic field guide may besubstantially parallel to a longitudinal axis of the magnetic fieldsource. In embodiments with one magnetic field source, the magneticfield source may have one or more magnetic field guides associated withthe magnetic field source. For example, the magnetic field source mayhave a first magnetic field guide and a second magnetic field guideassociated with the magnetic field source. In some embodiments, thedevice includes a first magnetic field guide disposed on a first surfaceof the magnetic field source, and a second magnetic field guide disposedon a second surface of the same magnetic field source. In someinstances, the first and second magnetic field guides are disposed onopposite surfaces of the magnetic field source. In certain embodiments,the first magnetic field guide is wedge-shaped with a first apex edge,the second magnetic field guide is wedge-shaped with a second apex edge,and the first apex edge is aligned substantially across from andparallel to the second apex edge. The first apex edge may be positionedat a substantially uniform distance along its length from the secondapex edge. In some cases, the magnetic field source includes a permanentmagnet, as described above, and the first and second surfaces of themagnetic field source are the north and south poles of the magneticfield source.

In embodiments with more than one magnetic field source, each magneticfield source may have a magnetic field guide associated with it. Eachmagnetic field guide may be positioned such that the longitudinal axisof the magnetic field guide is substantially parallel to thelongitudinal axis of the magnetic field source to which it isassociated.

In certain embodiments, the apex edge of the magnetic field guide has alinear profile. By “linear” is meant that the apex edge of the magneticfield guide is substantially straight. In some instances, the apex edgeof the magnetic field guide has a non-linear profile, such as, but notlimited to, a saw-tooth, sinusoidal, square wave, triangular waveprofile, combinations thereof, and the like. A magnetic field guide thathas an apex edge with a non-linear profile may facilitate a localincrease in the magnetic field and/or the magnetic field gradient nearthe non-linear portions of the apex edge.

The magnetic field guide may be proximal to the magnetic field source.In certain cases, the magnetic field guide is contacted with themagnetic field source. For example, the magnetic field guide may beattached to the magnetic field source to facilitate contact between themagnetic field guide and the magnetic field source. As described above,the device may include one magnetic field source. In these embodiments,the magnetic field source may include a wedge-shaped portion asdescribed above. The magnetic field source may also include an extendedportion between the wedge-shaped portion and the magnetic field source.The extended portion of the magnetic field guide may be configured toposition the wedge-shaped portion at a distance away from the surface ofthe magnetic field source. For example, the extended portion of themagnetic field guide may contact the magnetic field source on a part ofa first surface of the extended portion of the magnetic field guide. Theextended portion of the magnetic field guide may extend a distance abovethe top surface of the magnetic field source. The part of the firstsurface of the extended portion of the magnetic field guide that extendsabove the top surface of the magnetic field source may have thewedge-shaped portion of the magnetic field guide. In some embodiments,the extended portion and the wedge-shaped portion of the magnetic fieldguide are contiguous (e.g., formed from the same piece of material). Inother cases, the extended portion and the wedge-shaped portion of themagnetic field guide are separate pieces that are attached to eachother. As described above, the device may also include a second magneticfield guide disposed on a surface of the magnetic field source oppositefrom the first magnetic field guide. Similar to the first magnetic fieldguide described above, the second magnetic field guide may include anextended portion and a wedge-shaped portion. The first and secondmagnetic field guides may be configured such that the apex edge of thewedge-shaped portion of the first magnetic field guide is proximal tothe apex edge of the wedge-shaped portion of the second magnetic fieldguide. In some cases, the apex edge of the first magnetic field guide issubstantially parallel to the apex edge of the second magnetic fieldguide. The apex edge of the first magnetic field guide may be alignedacross from the apex edge of the second magnetic field guide. Forexample, the apex edge of the first magnetic field guide may be alignedsubstantially directly across from the apex edge of the second magneticfield guide. In certain embodiments, the apex edge of the first magneticfield guide is aligned substantially across from and substantiallyparallel to the apex edge of the second magnetic field guide. Duringuse, the distance between the apex edge of the first magnetic fieldguide and the apex edge of the second magnetic field guide may be 5 cmor less, such as 2 cm or less, including 1 cm or less, or 7 mm or less,or 5 mm or less, or 3 mm or less, or 2 mm or less, or 1 mm or less.

In other embodiments as described above, the device may include twomagnetic field sources, such as first and second magnetic field sourcesarranged proximal to each other. In some instances, a first magneticfield guide is associated with the first magnetic field source, and asecond magnetic field guide is associated with the second magnetic fieldsource. The first magnetic field guide may be positioned on the firstmagnetic field source on the surface of the first magnetic field sourceproximal to the second magnetic field source. For example, inembodiments where the magnetic field guides are wedge-shaped, the firstmagnetic field guide may be disposed on the first magnetic field sourcesuch that the base of the first magnetic field guide contacts thesurface of the first magnetic source proximal to the second magneticsource. Similarly, the second magnetic field guide may be positioned onthe second magnetic field source on the surface of the second magneticfield source proximal to the first magnetic field source. For example,in embodiments where the magnetic field guides are wedge-shaped, thesecond magnetic field guide may be disposed on the second magnetic fieldsource such that the base of the second magnetic field guide contactsthe surface of the second magnetic source proximal to the first magneticsource. In this arrangement, the first and second magnetic field guidesmay be positioned between the first and second magnetic field sources.In addition, the apex edge of the first magnetic field guide may beproximal to the apex edge of the second magnetic field guide. In somecases, the apex edge of the first magnetic field guide is substantiallyparallel to the apex edge of the second magnetic field guide. The apexedge of the first magnetic field guide may be aligned across from theapex edge of the second magnetic field guide. For example, the apex edgeof the first magnetic field guide may be aligned substantially directlyacross from the apex edge of the second magnetic field guide. In certainembodiments, the apex edge of the first magnetic field guide is alignedsubstantially across from and substantially parallel to the apex edge ofthe second magnetic field guide. During use, the distance between theapex edge of the first magnetic field guide and the apex edge of thesecond magnetic field guide may be 5 cm or less, such as 2 cm or less,including 1 cm or less, or 7 mm or less, or 5 mm or less, or 3 mm orless, or 2 mm or less, or 1 mm or less.

As described above, the first and second magnetic field guides may beconfigured to focus the magnetic field produced by the magnetic fieldsource. In certain instances, the first and second magnetic field guidesfocus the magnetic field to a region proximal to the apex edges of thefirst and second magnetic field guides. For example, the first andsecond magnetic field guides may focus the magnetic field in an areabetween the apex edges of the magnetic field guides. The first andsecond magnetic field guides may be configured to produce a magneticflux proximal to the apex edges of the magnetic field guides sufficientto separate magnetically labeled moieties from non-magnetically labeledmoieties in a sample. In some instances, the first and second magneticfield guides are configured to produce a magnetic field proximal to theapex edges of the magnetic field guides having a magnetic flux densityranging from 0.01 T to 10 T, or from 0.01 T to 5 T, or from 0.01 T to 2T, such as from 0.1 T to 2 T, including from 0.5 T to 1.5 T.

In certain embodiments, the magnetic field guide includes a soft magnet.The term “soft magnet” refers to a material that can be magnetized inthe presence of an applied external magnetic field, but whose magnetismsubstantially decreases when the external magnetic field is removed.Soft magnets may include, but are not limited to, ferromagneticmaterials, such as iron (e.g., annealed iron), stainless steel andnickel, ferrimagnetic materials, such as ceramic oxides of metals,combinations thereof, and the like.

In some instances, the magnetic field guide may have a length rangingfrom 1 cm to 100 cm, such as from 1 cm to 75 cm, including from 1 cm to50 cm, or from 1 cm to 25 cm, or from 1 cm to 10 cm, or from 5 cm to 10cm, for example from 5 cm to 6 cm; a width ranging from 0.1 cm to 100cm, such as from 0.1 cm to 75 cm, including from 0.1 cm to 50 cm, orfrom 0.1 cm to 25 cm, or form 0.1 cm to 10 cm, or from 0.1 cm to 5 cm,or from 0.1 cm to 2 cm, or from 0.5 cm to 2 cm, for example from 1 cm to1.5 cm; and a height ranging from 0.1 cm to 100 cm, such as from 0.1 cmto 75 cm, including from 0.1 cm to 50 cm, or from 0.1 cm to 25 cm, orfrom 0.1 cm to 10 cm, or from 0.1 cm to 5 cm, or from 0.1 cm to 2 cm, orfrom 0.5 cm to 2 cm, for example from 1 cm to 1.5 cm.

An example of an embodiment of a magnetic separation device according tothe present disclosure is shown in the schematic illustrations in FIGS.1(a), 1(b) and 1(c). The device includes two soft magnetic field guides2. Each magnetic field guide 2 is attached to a permanent magnet 1. Thetwo soft magnetic field guides 2 have a tapered shape from the endattached to the permanent magnet 1 towards the apex edges of the twomagnetic field guides that are directly opposite each other. The apexedges of the magnetic field guides 2 are substantially linear, as shownin FIGS. 1(b) and 1(c). The permanent magnets 1 have magnetizations 12that are in the same direction and perpendicular to the interfacebetween the permanent magnets 1 and the magnetic field guides 2. Themagnetic field guides 2 and permanent magnets 1 form a permanent magnetdriven magnetic flux concentration structure, where the magnetic fluxfrom permanent magnets 1 is focused (e.g., increased) by the taperedshape of the magnetic field guides 2. The magnetic field guides 2produce a locally high magnetic flux density in the area proximal to theapex edges of the magnetic field guides. In certain instances, the highmagnetic flux produces a high magnetic field and magnetic field gradientin the area proximal to, such as near and/or between, the apex edges ofthe magnetic field guides.

Another embodiment of the magnetic separation device is shown in FIGS.4(a) and 4(b). As shown in FIG. 4(a), the arrangement of the magneticfield sources (e.g., permanent magnets) 1 and the magnetic field guides2 is the same as in FIG. 1(a). However, as shown in FIG. 4(b), ratherthan having a linear profile, the magnetic field guides 2 have apexedges with a saw-tooth profile. In certain embodiments, the cornersalong the saw-tooth apex edge have a locally enhanced magnetic field andmagnetic field gradient, which may facilitate the separation of magneticlabels and magnetically labeled moieties from non-magnetically labeledmoieties in the sample.

Another embodiment of a magnetic separation device is shown in theschematic illustrations in FIGS. 8(a) and 8(b). The device includes twosoft magnetic field guides 2. The magnetic field guides 2 are attachedto opposing sides of the same permanent magnet 1. The magnetic fieldguides 2 each have a wedge-shaped portion with a tapered shape. Thewedge-shaped portions of the magnetic field guides 2 havecross-sectional areas that decrease towards the apex edges of themagnetic field guides. The apex edges of the magnetic field guides 2 aresubstantially linear and positioned directly opposite each other. Thepermanent magnet 1 has a magnetization 12 that is perpendicular to theinterfaces between the permanent magnet 1 and the magnetic field guides2. The magnetic field guides 2 and permanent magnet 1 form a permanentmagnet driven magnetic flux concentration structure, where the magneticflux from permanent magnet 1 is focused (e.g., increased) by the taperedshape of the wedge-shaped portions of the magnetic field guides 2. Themagnetic field guides 2 produce a locally high magnetic flux density inthe area proximal to (e.g., near and/or between) the apex edges of themagnetic field guides. In certain instances, the high magnetic fluxproduces a high magnetic field and magnetic field gradient in the areaproximal to the apex edges of the magnetic field guides.

In certain embodiments, the device includes one or more magnetic fluxsinks. The magnetic flux sink may be disposed on a surface of themagnetic field source. In some instances, the magnetic flux sink isdisposed on a surface of the magnetic field source opposite the surfaceof the magnetic field source in contact with the magnetic field guide.In certain cases, the magnetic flux sink is configured to increase themagnetic field of the magnetic field source. The magnetic flux sink maybe configured to increase the magnetic field of the magnetic fieldsource by decreasing the self-demagnetization field of the magneticfield source (e.g., the self-demagnetization field of the permanentmagnet). In some cases, the magnetic flux sink includes a soft magnet.

Conduit

Embodiments of the device for separating magnetically labeled moietiesin a sample may further include a conduit. The conduit may be configuredto direct a flow of the sample through the device. As such, the conduitmay be configured to carry the flow of the sample (e.g., a samplesolution) in a channel, tube, well, etc. In certain embodiments, theconduit is enclosed, such that the conduit is defined by outer wallsthat surround a central flow path. The central flow path may be alignedwith a longitudinal axis of the conduit. The central flow path may haveany convenient shape, such as, but not limited to, a flow path with across-sectional profile of a circle, an ellipse, a square, a rectangle,a pentagon, a hexagon, an irregular cross-sectional profile,combinations thereof, and the like. During use, the conduit may also beconfigured to retain the magnetically labeled moieties in the sample.

In some instances, at least a portion of the conduit is positionedbetween the magnetic field guides, such as between the first magneticfield guide and the second magnetic field guide. The conduit may bepositioned between the first and second magnetic field guides such thata longitudinal axis of the conduit is substantially parallel to alongitudinal axis of the first magnetic field guide and a longitudinalaxis of the second magnetic field guide. For example, the conduit may bepositioned between the apex edges of the first and second magnetic fieldguides such that the longitudinal axis of the conduit is substantiallyparallel to the apex edges of each of the first and second magneticfield guides. In some cases, positioning the conduit substantiallyparallel to the apex edges of the magnetic field guides maximizes thelength of conduit, and thus the flow of sample fluid, that is betweenthe apex edges of the magnetic field guides. In certain instances,positioning the conduit substantially parallel to the apex edges of themagnetic field guides maximizes the amount of time the flow of thesample is between the magnetic field guides. Aligning the conduitsubstantially parallel to the apex edges of the magnetic field guidesmay facilitate retaining the magnetically labeled moieties in theconduit.

In some instances, at least a portion of the conduit is positionedproximal to the magnetic field guides, such as adjacent the firstmagnetic field guide and the second magnetic field guide. In someinstances, the conduit is positioned adjacent to, but not between, theapex edges of the first and second magnetic field guides. In certaincases, the conduit is positioned such that the conduit is in directcontact with an outer surface of one or more of the magnetic fieldguides. For example, the conduit may be positioned such that the conduitcontacts the angled outer surface of the wedged-shaped portion of themagnetic field guides. In some cases, the conduit may not be positioneddirectly between the apex edges of the magnetic field guides, but ratheradjacent to the apex edges and contacting an outer surface of themagnetic field guides as described above. The conduit may be positionedproximal to the first and second magnetic field guides such that alongitudinal axis of the conduit is substantially parallel to alongitudinal axis of the first magnetic field guide and a longitudinalaxis of the second magnetic field guide. For example, the conduit may bepositioned adjacent to the first and second magnetic field guides suchthat the longitudinal axis of the conduit is substantially parallel tothe apex edges of each of the first and second magnetic field guides. Insome cases, positioning the conduit substantially parallel to the apexedges of the magnetic field guides maximizes the length of conduit, andthus the flow of sample fluid, that is adjacent to the apex edges of themagnetic field guides. In certain instances, positioning the conduitsubstantially parallel to the apex edges of the magnetic field guidesmaximizes the amount of time the flow of the sample is proximal to themagnetic field guides. Aligning the conduit substantially parallel tothe apex edges of the magnetic field guides may facilitate retaining themagnetically labeled moieties in the conduit.

In some instances, the conduit is configured to have a narrowercross-sectional area in the portion of the conduit positioned betweenthe magnetic field guides. For example, the cross-sectional area of theconduit upstream from the portion of the conduit positioned between themagnetic field guides may be greater than the cross-sectional area ofthe portion of the conduit positioned between the magnetic field guides.Similarly, the cross-sectional area of the conduit downstream from theportion of the conduit positioned between the magnetic field guides maybe greater than the cross-sectional area of the portion of the conduitpositioned between the magnetic field guides. Thus, in some cases, aportion of the conduit positioned between the first and second magneticfield guides has a cross-sectional area less than the cross-sectionalarea of a portion of the conduit upstream or downstream from the portionof the conduit positioned between the first and second magnetic fieldguides.

In certain embodiments, the conduit may be positioned between themagnetic field guides manually. For example, the conduit may be manuallyaligned between the magnetic field guides, and may be manually removedfrom between the magnetic field guides. The conduit may be configured tohave one or more alignment guides on the exterior of the conduit, suchas, but not limited to, a notch, a tab, a groove, a guide post, etc.,which may facilitate positioning of the conduit between the magneticfield guides. In some embodiments, the device may be configured toautomatically position the conduit between the magnetic field guides.The conduit may include one or more markings or alignment guides asdescribed above that the device may use to position the conduit betweenthe magnetic field guides.

In some instances, the conduit is configured to be positionable awayfrom the magnetic field, e.g., positionable away from the magnetic fieldsources and the magnetic field guides. Positioning the conduit away fromthe magnetic field may facilitate the recovery of magnetically labeledmoieties that were retained in the conduit during an assay. In certaincases, the device may be configured to automatically position theconduit away from the magnetic field guides.

In certain cases, the conduit is configured to be reusable. A reusableconduit may be configured to be washed between assays, such as, but notlimited to, configured to be washed by flowing a wash solution or bufferthrough the conduit between assays. In some cases, the conduit may beconfigured to be washed and reused without removing the conduit from thedevice. In other cases, the conduit may be configured to be removed fromthe device, washed and then reinserted into the device for a subsequentassay. In certain embodiments, the conduit is configured to bedisposable. By disposable is meant that the conduit may be used once orseveral times (e.g., 20 times or less, 15 times or less, 10 times orless, or 5 times or less) and then discarded and replaced by a newconduit. For example, the conduit may be configured to be a single-useconduit, where the conduit is configured to be used for a single assay,and then removed and discarded. A new conduit may be used in asubsequent assay.

In certain embodiments, the conduit may have a height (e.g., forconduits that do not have a round cross-sectional profile) or an innerdiameter (e.g., for conduits that have a round cross-sectional profile)of 5 cm or less, such as 2 cm or less, including 1 cm or less, or 7 mmor less, or 5 mm or less, or 3 mm or less, or 2 mm or less, or 1 mm orless. The length of the conduit may range from 1 cm to 1000 cm, such asfrom 2 cm to 750 cm, including from 5 cm to 500 cm, or from 5 cm to 250cm, or from 10 cm to 100 cm, such as from 10 cm to 50 cm, for examplefrom 10 cm to 25 cm.

In certain embodiments, the conduit is configured to be substantiallyfree from magnetic gradient enhancing materials. For example, theconduit may be made of non-magnetic and/or non-magnetizable materials.In some instances, the central flow path of the conduit is substantiallyfree from magnetic gradient enhancing materials (excluding the magneticlabels themselves). For instance, the central flow path of the conduitmay be substantially free of any materials (e.g., matrix materials,magnetizable particles (e.g., magnetizable spheres/ellipsoids),magnetizable wires, magnetizable cylinders, and the like) other than thesample (e.g., including any buffer and magnetic labels, etc. used in theassay itself). In some instances, having a conduit with a central flowpath substantially free of materials, such as magnetizable materials,facilitates the subsequent recovery of the separated magneticallylabeled moieties. For example, the separated magnetically labeledmoieties may be more easily flushed from the conduit when the conduit issubstantially free of materials as compared to a conduit with materials,such as magnetizable materials, in the central flow path of the conduit.The separated magnetically labeled moieties may be more easily flushedfrom the conduit, for instance, due to the absence of restrictions tothe fluid flow path in a conduit substantially free of materials and/orthe absence of magnetizable materials in the flow path that may haveremnant magnetizations that retain the magnetically labeled moieties inthe conduit.

In certain embodiments, the conduit includes a material that isflexible. When positioned between the magnetic field guides, themagnetic field guides, in some instances, may contact the surface of theconduit. In some cases, the first magnetic field guide (e.g., the apexedge of the first magnetic field guide) contacts a surface of theconduit, and the second magnetic field guide (e.g., the apex edge of thesecond magnetic field guide) contacts an opposing surface of theconduit. The magnetic field guides may be configured to contact thesurfaces of the conduit without exerting significant pressure on theconduit. In other embodiments, the device is configured to compress theconduit between the apex edge of the first magnetic field guide and theapex edge of the second magnetic field guide. In some instances, theconduit is compressed such that the height (e.g., inner diameter) of theconduit is compressed to a fraction of the height of the conduit in theabsence of any compression. For example, the conduit may be compressedto 90% or less of its initial height, such as 80% or less, including 70%or less, or 60% or less, or 50% or less of its initial height. Incertain embodiments, the conduit is configured such that the conduit maybe compressed near the center of the conduit, but may retainsubstantially the same height towards the outer edges of the conduit. Inthese embodiments, under compression, the conduit may have a centralflow path with a height less than the height of the flow path near theouter edges of the conduit. Having a central flow path with a heightless than the height of the flow path near the outer edges of theconduit may facilitate the retention of the magnetically labeledmoieties in the conduit because the flow rate through the narrowercenter flow path of the conduit may be less than the flow rate throughthe wider periphery of the conduit.

The conduit may be made of any material that is compatible with theassay conditions, e.g., the sample solution buffer, pressure,temperature, etc. For example, the conduit may include materials thatare substantially non-reactive to the sample, the moieties in thesample, the buffer, and the like. The conduit may include a flexiblematerial, such that the conduit is flexible. In certain instances, theconduit is configured to deform from its initial shape and/or stretch ifthe conduit is compressed between the apex edges of the magnetic fieldguides, as described above. The conduit may be configured to deform fromits initial shape and/or stretch without breaking, splitting, tearing,etc., when the conduit is compressed between the magnetic field guides.In some instances, the conduit includes glass, or polymers, such as, butnot limited to, silicone, polypropylene, polyethylene, polyether etherketone (PEEK), and the like. In certain embodiments, the conduitincludes a flexible material, such as a flexible polymer material (e.g.,silicone, polyethylene, polypropylene, PEEK, etc.).

In some instances, the conduit has a cover layer disposed on the outersurface of the conduit. The cover layer may be configured to protect theconduit from the surrounding environment, and in some instances, mayinclude one or more alignment guides to facilitate positioning theconduit between the magnetic field guides, as described above. The coverlayer may include a flexible material, such that the cover layer isflexible and may deform from its initial shape and/or stretch. Incertain instances, the cover layer is configured to deform from itsinitial shape and/or stretch if the conduit is compressed between theapex edges of the magnetic field guides as described above. The coverlayer may be configured to deform from its initial shape and/or stretchwithout breaking, splitting, tearing, etc., when the conduit iscompressed between the magnetic field guides. In certain embodiments,the conduit includes a flexible material, such as a flexible polymermaterial (e.g., silicone, polyethylene, polypropylene, PEEK, etc.).

An example of a conduit according to embodiments of the presentdisclosure is shown in FIGS. 2(a) and 2(b). The conduit 20 has a centralflow path 22 configured to carry a flow of a sample through the device.The conduit may be configured with a rectangular cross-sectional profile(see FIG. 2(b)). In some instances, a conduit with a rectangularcross-sectional profile may facilitate alignment of the conduit betweenthe magnetic field guides of the device.

FIGS. 3(a) and 3(b) show schematics of front views of a conduitpositioned between the magnetic field guides in a magnetic separationdevice, according to embodiments of the present disclosure. The conduit3 is positioned within the gap between the opposing apex edges of themagnetic field guides as shown in FIGS. 3(a) and 3(b). A liquid samplewith magnetically labeled biological or chemical moieties flows withinthe conduit 3 and along the tapered apex edges of the magnetic fieldguides. The magnetic field and magnetic field gradient produced by themagnetic field sources attracts the magnetic labels and magneticallylabeled moieties from the flowing sample. The magnetic labels andmagnetically labeled moieties are then pulled to and are retained at theinner surface of the conduit proximal to the apex edges of the magneticfield guides. Thus, magnetic labels and magnetically labeled moietiesare separated from the flowing solution and retained within the conduit.After the solution sample is flowed through the conduit and a pluralityof magnetic labels and magnetically labeled moieties are separated fromthe flowing solution, conduit 3 is then removed from the gap between themagnetic field guides 2 and the magnetic field within the conduitbecomes approximately zero. By flushing the retained magnetic labels andmagnetically labeled moieties within the conduit from the conduit with abuffer solution, the magnetic labels and magnetically labeled moietiescan then be recovered from the conduit.

FIG. 3(a) shows an embodiment where the conduit 3 has a rectangularcenter flow path, where the magnetic labels and magnetically labeledmoieties are retained towards center of the conduit. FIG. 3(b) shows anembodiment where the conduit has a pinched center flow path (e.g., thecenter flow path has a height less than the height of the flow path nearthe periphery of the flow path). In FIG. 3(b), magnetic labels andmagnetically labeled moieties are retained near the pinched centerportion of the flow path. In certain instances, because the side areasof the conduit have a larger clearance height than the pinched centerflow path, when solution flows within the conduit, the solution flowingthrough the center part of the conduit experiences a slower flow ratethan solution flowing through the side areas. Thus, in some cases, themagnetic labels and magnetically labeled moieties experience less flowsheer force than in the embodiment shown in FIG. 3(a), which mayfacilitate magnetic separation efficiency.

FIGS. 6(a), 6(b) and 6(c) show graphs of a simulated magnetic field(FIG. 6(a)), magnetic field gradient (FIG. 6(b)), and product of themagnetic field and absolute magnetic field gradient (FIG. 6(c)) acrossthe gap between the magnetic field guides as shown in FIG. 1(a) for amagnetic separation device with a distance between the apex edges of themagnetic field guides of 1.4 mm, according to embodiments of the presentdisclosure. The x-axis is along the center of the gap from left to rightbetween the apex edges of the magnetic field guides, as shown in FIG.3(a). FIG. 6(a) shows that a magnetic field of 1.4 Tesla or more can beachieved in certain embodiments. FIG. 6(b) shows a graph of the magneticfield gradient calculated from the magnetic field profile shown in FIG.6(a), where the gradient peak is 0.8 T/mm or more across the gap betweenthe apex edges of the magnetic field guides. The gradient valuesindicate a strong magnetic force on the magnetic labels towards the apexedges of the magnetic field guides. FIG. 6(c) shows a graph of theproduct of the magnetic field and absolute magnetic field gradient,which is proportional to the magnetic force on the magnetically labeledmoieties flowing through the conduit. In some instances, themagnetically labeled moieties within the conduit are attracted andretained within +/−0.5 mm of the apex edge of the magnetic field guide.

FIG. 5 illustrates another embodiment of a conduit positioned in amagnetic separation device according to embodiments of the presentdisclosure. FIG. 5 shows a schematic of a magnetic separation device 50with a conduit 52 having a flow path 58 positioned between the magneticfield guides 54 and the magnetic field sources (e.g., permanent magnets)56. The conduit flow path 58 shown in FIG. 5 has a taperedcross-sectional shape such that the cross-sectional dimension of theconduit proximal to the apex edges of the magnetic field guides is lessthan the cross-sectional dimension distal to the apex. This allows theapexes of to be positioned closer together, and, further, facilitatespositioning of the conduit 52 between the apex edges of the magneticfield guides 54.

Conduit Holder

In certain embodiments, the device includes a conduit holder operativelycoupled to the conduit. In some cases, the conduit holder is configuredto operatively couple the conduit to the magnetic separation device. Forexample, the conduit holder may be configured to facilitate positioningof the conduit between the magnetic field guides. In some cases, theconduit holder includes an elongated tab attached to the exterior of theconduit. The elongated tab may be attached to the exterior of theconduit such that the elongated tab is substantially parallel to alongitudinal axis of the conduit. In certain instances, the conduitholder facilitates positioning the conduit in the magnetic separationdevice such that a longitudinal axis of the conduit is substantiallyparallel to a longitudinal axis of the magnetic separation device, suchas substantially parallel to the apex edges of the magnetic field guidesas described above.

In some cases, the magnetic separation is configured to mate with theconduit holder operatively coupled to the conduit. For example, themagnetic separation device may be configured to have one or more matingelements, such as, but not limited to, a notch, a tab, a groove, achannel, a guide post, etc., which correspond to one or morecorresponding alignment guides on the conduit holder. The one or moremating elements may facilitate positioning the conduit between themagnetic field guides of the magnetic separation device. In some cases,the magnetic separation device includes a channel configured to matewith the conduit holder. The channel may be configured to position theconduit holder in the magnetic separation device such that thelongitudinal axis of the conduit is substantially parallel to alongitudinal axis of the magnetic separation device, such assubstantially parallel to the apex edges of the magnetic field guides asdescribed above.

In certain embodiments, the conduit holder may be positioned between themagnetic field guides manually. For example, the conduit holder may bemanually positioned in the magnetic separation device by aligning theconduit holder with the corresponding mating element (e.g., channel) ofthe magnetic separation device. In some cases, the conduit holder may bemanually removed from the magnetic separation device. In someembodiments, the device may be configured to automatically position theconduit holder in the magnetic separation device. The conduit holder mayinclude one or more markings or alignment guides as described above thatthe device may use to automatically position the conduit holder in themagnetic separation device.

FIG. 9 shows a schematic cross-section of a conduit positioned in amagnetic separation device. Fluid sample flows through conduit 901,which is operatively coupled to a conduit holder 902 and positioned in amagnetic separation device and in close proximity to the apex edges ofthe magnetic field guides 2. The conduit 901 is a tubing, which can beflexible or rigid, and is positioned with its outside surface in contactwith the magnetic field guides 2, but not between the apex edges of themagnetic field guides, as shown in FIG. 9. Conduit holder 902facilitates positioning the conduit 901 in the magnetic separationdevice such that a longitudinal axis of the conduit 901 is substantiallyparallel to the longitudinal axes of the magnetic field guides 2.

FIG. 10(a) is a front view of a conduit 1001 operatively coupled to aconduit holder 1002. FIG. 10(b) is a three-dimensional schematic of theconduit 1001 operatively coupled to the conduit holder 1002. The conduit1001 has a central flow path that carries the flow of the sample fluidthrough the magnetic separation device. The conduit 1001 also includesalso includes two openings 1003 and 1004, which may be connected to afluid reservoir, a fluid transfer connector or an adapter, as desired.

FIG. 11 is a three-dimensional perspective view of the conduit 1101operatively coupled to a conduit holder 1102 positioned in a magneticseparation device 1103. The conduit 1101 is positioned above the gapbetween the opposing apex edges of the magnetic field guides as shown inFIG. 9. A liquid sample with magnetically labeled biological or chemicalmoieties flows within the conduit 1101 and adjacent the tapered apexedges of the magnetic field guides. The magnetic field and magneticfield gradient produced by the magnetic field sources attracts themagnetic labels and magnetically labeled moieties in the flowing sample.The magnetic labels and magnetically labeled components are then pulledto and retained at the inner surface of the conduit proximal to the apexedges of the magnetic field guides. Thus, magnetic labels andmagnetically labeled moieties are separated from the flowing solutionand retained within the conduit. After the sample solution is flowedthrough the conduit and a plurality of magnetic labels and magneticallylabeled moieties are separated from the flowing solution, the conduit isthen positioned away from the magnetic field guides and the magneticfield within the conduit becomes approximately zero. By flushing theretained magnetic labels and magnetically labeled moieties from theconduit with a solution (e.g., a buffer solution), the magnetic labelsand magnetically labeled moieties can be recovered from the conduit.

Magnetic Labels

Magnetic labels are labeling moieties that are retained by the devicefor separating magnetically labeled moieties in a sample. Magneticlabels of interest may be retained by the device if they flow through aportion of a conduit in close proximity to the magnetic field producedby the device, e.g., between the magnetic field sources and/or betweenthe magnetic field guides of the device).

Magnetic labels useful in the practice of certain embodiments of thepresent disclosure are magnetic particles, such as, but not limited toferromagnetic, paramagnetic, super-paramagnetic, anti-ferromagnetic, orferrimagnetic particles. In certain instances, the magnetic particlesappear “nonmagnetic” (e.g., have a remnant magnetization ofsubstantially zero) in the absence of a magnetic field. Magneticparticles with a substantially zero remnant magnetization may notsubstantially agglomerate with each other in solution in the absence ofan external magnetic field.

The magnetic particles may be chemically stable in a biologicalenvironment, which may facilitate their use in the assay conditions. Insome cases, the magnetic particles are biocompatible, e.g., watersoluble and functionalized so that they may be readily attached tobiomolecules of interest, such as an antibody that specifically binds toa target analyte. By associating or binding magnetic particles to aspecific antibody, the magnetic particles may be targeted to a specificanalyte through the specific binding interactions between the antibodyand complementary antigen. In some instances, the magnetic label may bebound to the protein or antibody as described above through anon-covalent or a covalent bond with each other. Examples ofnon-covalent associations include non-specific adsorption, binding basedon electrostatic (e.g., ion, ion pair interactions), hydrophobicinteractions, hydrogen bonding interactions, specific binding through aspecific binding pair member covalently attached to the surface of themagnetic particle, and the like. Examples of covalent binding includecovalent bonds formed between the biomolecule and a functional grouppresent on the surface of the magnetic particle, e.g. —OH, where thefunctional group may be naturally occurring or present as a member of anintroduced linking group.

In certain embodiments, the magnetic particles are nanoparticles. By“nanoparticle” is meant a particle having an average size (e.g., meandiameter) in the range of 1 nm to 1000 nm. In certain embodiments, theaverage size (e.g., mean diameter) of the magnetic nanoparticles issub-micron sized, e.g., from 1 nm to 1000 nm, or from 1 nm to 500 nm, orfrom 5 nm to 250 nm, such as from 5 nm to 150 nm, including from 5 nm to50 nm. For example, magnetic nanoparticles having a mean diameter of 5nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50nm, 55 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm,140 nm, 150 nm, and 200 nm as well as nanoparticles having meandiameters in ranges between any two of these values, are suitable foruse herein. In certain embodiments, the magnetic particles aresubstantially uniform in shape. For example, the magnetic particles maybe spherical in shape. In addition to a spherical shape, magneticnanoparticles suitable for use herein can be shaped as disks, rods,coils, fibers, pyramids, and the like.

Methods

Aspects of the present disclosure include methods of separatingmagnetically labeled moieties in a sample. The magnetically labeledmoieties may be separated from the other components of the sample, suchas non-magnetically labeled moieties (e.g., moieties that are notassociated with a magnetic label).

In certain embodiments, the method includes positioning a conduit in amagnetic separation device as described above. The positioning may beperformed manually or automatically. In embodiments where thepositioning is performed manually, the user may position the conduit inthe device such that the conduit is aligned proximal to (e.g., adjacentto or between) the magnetic field guides of the device. For instance,positioning the conduit may include aligning the conduit such that alongitudinal axis of the conduit is substantially parallel to alongitudinal axis of the magnetic field guides (e.g., the longitudinalaxis of the first magnetic field guide and the longitudinal axis of thesecond magnetic field guide). In embodiments where the positioning isperformed automatically by the device, the device may be programmed toposition the conduit proximal to (e.g., adjacent to or between) themagnetic field guides without the intervention of the user. For example,the device may automatically align the conduit such that thelongitudinal axis of the conduit is substantially parallel to thelongitudinal axis of the magnetic field guides (e.g., the longitudinalaxis of the first magnetic field guide and the longitudinal axis of thesecond magnetic field guide).

Aspects of the method further include applying a magnetic field to thesample. In some instances, the sample is a sample solution flowingthrough the conduit, thus the method includes applying a magnetic fieldto the sample flowing through the conduit. In certain instances, themethod includes applying a magnetic field having a magnetic fluxsufficient to separate magnetically labeled moieties fromnon-magnetically labeled moieties in the sample. The magnetic field maybe applied continuously as the sample flows through the conduit, or maybe applied discontinuously in a pulsed application. In certainembodiments, the magnetic field source is a permanent magnet asdescribed above, and thus the magnetic field is applied continuously tothe sample as the sample flows through the conduit.

In certain embodiments, the method includes positioning the conduit awayfrom the magnetic field. The conduit may be positioned away from themagnetic field such that the applied external field on the conduit issubstantially zero. Positioning the conduit away from the magnetic fieldmay be achieved by removing the conduit from the device. For instance,the conduit may be removed from its position proximal to (e.g., adjacentto or between) the magnetic field guides and moved to a position awayfrom the magnetic field source and the magnetic field guides.Positioning the conduit away from the magnetic field may facilitate thesubsequent recovery of any magnetically labeled moieties that wereretained in the conduit during the assay. In certain instances,positioning the conduit away from the magnetic field may be performedmanually, while in other embodiments, positioning the conduit away fromthe magnetic field may be performed automatically (e.g., without theintervention of the user).

In some instances, positioning the conduit away from the magnetic fieldmay be achieved by moving the magnetic field source (and the associatedmagnetic field guides) away from the conduit. For example, inembodiments that include one magnetic field source, the magnetic fieldsource and the associated magnetic field guides may be moved to aposition away from the conduit such that the magnetic field guides donot produce a magnetic field having sufficient magnetic field strength,gradient strength, and/or magnetic flux to retain the magneticallylabeled moieties in the conduit.

In embodiments that include two magnetic field sources, the firstmagnetic field source and the second magnetic field source may be movedto positions away from the conduit. The magnetic field sources may bemoved such that the distance between the magnetic field sources isgreater than the distance between the magnetic field sources during theassay. For instance, the magnetic field sources may be moved topositions away from the conduit such that the distance between the apexedges of the magnetic field guides is greater than the distance betweenthe apex edges of the magnetic field guides during the assay. In somecases, the magnetic field sources may be moved to positions away fromthe conduit such that the magnetic field guides do not produce amagnetic field having sufficient magnetic field strength, gradientstrength, and/or magnetic flux to retain the magnetically labeledmoieties in the conduit.

Positioning the conduit away from the magnetic field may facilitate thesubsequent recovery of any magnetically labeled moieties that wereretained in the conduit during the assay. For example, after positioningthe conduit away from the magnetic field, the magnetically labeledmoieties retained in the conduit may be recovered by flushing themagnetically labeled moieties from the conduit. For instance, themagnetically labeled moieties may be recovered by flowing a buffer orother compatible solution through the conduit to flush (e.g., wash) themagnetically labeled moieties from the conduit. Alternatively, themagnetically labeled moieties may be recovered from the conduit bycentrifugation, application of a vacuum, pumping, combinations thereof,and the like.

Aspects of the methods disclosed herein may further includeconcentrating the recovered magnetically labeled moieties. Afterperforming the magnetic separation assay as described herein, themagnetically labeled moieties that were retained in the conduit duringthe magnetic separation assay may be recovered from the conduit byflushing the magnetically labeled moieties from the conduit as describedabove. In certain embodiments, it may be desirable to increase theconcentration of the magnetically labeled moieties in the solution thatis flushed from the conduit. Thus, the method may include concentrating(e.g., increasing the concentration of) the magnetically labeledmoieties in the solution that was flushed from the conduit.Concentrating the magnetically labeled moieties may include passing thesolution that was flushed from the conduit that contains themagnetically labeled moieties through a concentration device. Forexample, the concentration device may include, but is not limited to, anacoustic concentrator. Further description of acoustic concentrators isfound in U.S. Pat. No. 6,929,750, the disclosure of which is herebyincorporated by reference.

Assay methods disclosed herein may be qualitative or quantitative. Thus,as used herein, the term “detection” or “separation” refers to bothqualitative and quantitative determinations, and therefore includes“measuring” and “determining a level” of magnetically labeled moietiesin a sample.

Aspects of the methods disclosed herein may further include attaching amagnetic label to one or more target moieties in a sample prior toperforming the magnetic separation assay (e.g., prior to applying themagnetic field to the sample). As such, the method may includemagnetically labeling one or more moieties in a sample prior toperforming the magnetic separation assay. The magnetic label may bestably associated with the moiety (or moieties) of interest throughnon-covalent or covalent interactions as described above. For example,the magnetic label may be associated with the moiety of interest througha binding interaction between a binding pair of molecules.

The binding pair of molecules may vary depending on the bindinginteraction of interest. Binding interactions of interest include anyinteraction between the binding pair of molecules, where the bindinginteraction occurs with specificity between the binding pair ofmolecules under the environmental conditions of the binding interaction.Examples of binding interactions of interest include, but are notlimited to: nucleic acid hybridization interactions, protein-proteininteractions, protein-nucleic acid interactions, enzyme-substrateinteractions and receptor-ligand interactions, e.g., antibody-antigeninteractions and receptor-agonist or antagonist interactions.

Examples of molecules that have molecular binding interactions ofinterest include, but are not limited to: biopolymers and smallmolecules, which may be organic or inorganic small molecules. A“biopolymer” is a polymer of one or more types of repeating units.Biopolymers may be found in biological systems (although they may bemade synthetically) and may include peptides, polynucleotides, andpolysaccharides, as well as such compounds composed of or containingamino acid analogs or non-amino acid groups, or nucleotide analogs ornon-nucleotide groups. As such, biopolymers include polynucleotides inwhich the conventional backbone has been replaced with a non-naturallyoccurring or synthetic backbone, and nucleic acids (or synthetic ornaturally occurring analogs) in which one or more of the conventionalbases has been replaced with a group (natural or synthetic) capable ofparticipating in Watson-Crick type hydrogen bonding interactions. Forexample, a “biopolymer” may include DNA (including cDNA), RNA,oligonucleotides, PNA, other polynucleotides, and the like. A“biomonomer” references a single unit, which can be linked with the sameor other biomonomers to form a biopolymer (e.g., a single amino acid ornucleotide with two linking groups, one or both of which may haveremovable protecting groups).

In some instances, the binding pair of molecules are ligands andreceptors, where a given receptor or ligand may or may not be abiopolymer. The term “ligand” as used herein refers to a moiety that iscapable of covalently or otherwise chemically binding a compound ofinterest. Ligands may be naturally-occurring or manmade. Examples ofligands include, but are not restricted to, agonists and antagonists forcell membrane receptors, toxins and venoms, viral epitopes, hormones,opiates, steroids, peptides, enzyme substrates, cofactors, drugs,lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides,proteins, and the like. The term “receptor” as used herein is a moietythat has an affinity for a ligand. Receptors may be attached, covalentlyor non-covalently, to a binding member, either directly or via aspecific binding substance. Examples of receptors include, but are notrestricted to, antibodies, cell membrane receptors, monoclonalantibodies and antisera reactive with specific antigenic determinants,viruses, cells, drugs, polynucleotides, nucleic acids, peptides,cofactors, lectins, sugars, polysaccharides, cellular membranes,organelles, and the like. A “ligand receptor pair” is formed when twomolecules have combined through molecular recognition to form a complex.

Accordingly, the methods may include detecting a binding interactionbetween a binding pair of molecules. The binding interaction may includeone member of the binding pair of molecules that is labeled with amagnetic label as described herein. For example, one member of thebinding pair of molecules may be magnetically labeled and may bind toits complementary binding pair member to form a binding pair complex.The binding pair complex may be separated from the moieties not ofinterest in the sample using a magnetic separation device and methods asdescribed herein. After performing the magnetic separation assay, thebinding pair complex may be detected using any convenient method, suchas, but not limited to, flow cytometry, fluorescence detection,high-performance liquid chromatography (HPLC), electrophoresis,combinations thereof, and the like.

Aspects of methods of the present disclosure may further includeanalyzing the separated magnetically labeled moieties. In certaininstances, the magnetically labeled moieties are analyzed subsequent tobeing separated from the non-magnetically labeled moieties in thesample, as described above. As such, the method may include analyzingthe magnetically labeled moieties in the eluent from the magneticseparation device. In certain embodiments, the method includes analyzingthe magnetically labeled moieties to determine information about themagnetically labeled moieties. For example, analyzing the magneticallylabeled moieties may include counting the number of magnetically labeledmoieties that were retained by the magnetic separation device. In someinstances, the analyzing includes sorting the magnetically labeledmoieties. For instance, the method may include counting and/or sortingthe magnetically labeled moieties using a flow cytometry device. Incertain cases, analyzing the magnetically labeled moieties includesdetermining one or more physical and/or chemical properties of themagnetically labeled moieties, such as, but not limited to,fluorescence, mass, charge, chemical composition, UV absorption,infrared absorption, light scattering, combinations thereof, and thelike.

Systems

Systems according to embodiments of the present disclosure include oneor more magnetic separation devices for separating magnetically labeledmoieties in a sample. Each of the one or more magnetic separationdevices may be configured as described according to the presentdisclosure. For instance, the magnetic separation device includes amagnetic field source, a first magnetic field guide, and a secondmagnetic field guide as described herein. In addition, the systemincludes a conduit positioned proximal to the first magnetic field guideand the second magnetic field guide.

In certain embodiments, the system includes more than one magneticseparation device. For instance, the system may include 2 or moremagnetic separation devices, such as 3 or more, or 4 or more, or 5 ormore, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 ormore magnetic separation devices. The magnetic separation devices may bearranged in series such that the magnetic separation devices arepositioned upstream and downstream from each other in series. Arrangingthe magnetic separation devices in series may facilitate the progressiveseparation of magnetically labeled moieties from the same sample. Insome instances, the magnetic separation devices are arranged inparallel. Arranging the magnetic separation devices in parallel mayfacilitate the simultaneous separation of magnetically labeled moietiesfrom a plurality of samples. In certain cases, the magnetic separationdevices are arranged in series and in parallel.

In certain embodiments, the system includes more than one magneticseparation device, for example two magnetic separation devices (e.g., afirst magnetic separation device and a second magnetic separationdevice). The first and second magnetic separation devices may bearranged in series as described above. In some instances, the devicesmay be configured such that the apex edges of the first and secondmagnetic field guides of the first magnetic separation device havesubstantially the same profiles as the apex edges of the first andsecond magnetic field guides of the second magnetic separation device.For example, the apex edges of the first and second magnetic fieldguides of the first magnetic separation device and the apex edges of thefirst and second magnetic field guides of the second magnetic separationdevice may each have a linear profile (or any other profile as desired,as described above). In other embodiments, the devices may be configuredsuch that the apex edges of the first and second magnetic field guidesof the first magnetic separation device have different profiles from theapex edges of the first and second magnetic field guides of the secondmagnetic separation device. For example, the apex edges of the first andsecond magnetic field guides of the first magnetic separation device mayeach have a linear profile, and the apex edges of the first and secondmagnetic field guides of the second magnetic separation device may eachhave a saw-tooth profile. The above examples are for illustrativepurposes, and other combinations of profiles for the apex edges of themagnetic separation devices are also possible.

Embodiments of systems of the present disclosure may also include aconcentrator (e.g., a particle concentration device). The concentratormay be arranged downstream from the magnetic separation device. In someinstances, the concentrator is configured to increase the concentrationof magnetically labeled moieties in the eluent from the magneticconcentration device. The concentrator may be any type of concentrator,and in some embodiments is an acoustic concentrator.

Aspects of systems of the present disclosure may also include a particleanalysis device. The particle analysis device may be arranged downstreamfrom the magnetic separation device, and in certain instances may bearranged downstream from the concentrator. The particle analysis devicemay be configured to analyze the magnetically labeled moieties anddetermine information about the magnetically labeled moieties. Forexample, the particle analysis device may be configured to count thenumber of magnetically labeled moieties that were retained by themagnetic separation device. In some instances, the particle analysisdevice may be configured to sort the magnetically labeled moieties. Incertain cases, the particle analysis device may analyze the magneticallylabeled moieties to determine one or more physical and/or chemicalproperties of the magnetically labeled moieties, such as, but notlimited to, fluorescence, mass, charge, chemical composition, UVabsorption, infrared absorption, light scattering, combinations thereof,and the like. In certain embodiments, the particle analysis deviceincludes a flow cytometer, a mass spectrometer, an electrophoresisdevice, a high-performance liquid chromatography (HPLC) device, a UVspectrometer, an infrared spectrometer, and the like. In some instances,the particle analysis device is a flow cytometer.

Systems of the present disclosure may further include other supportdevices and/or additional components that may facilitate the performanceof the magnetic separation assay and/or any subsequent analysis of theseparated magnetically labeled moieties. For example, the system mayfurther include a computer programmed to perform the magnetic separationassay, fluid handling components configured to provide a flow of thesample solution and/or buffer through the system (e.g., a pump, a vacuumsource, a fluid reservoir, valves, inlets, outlets, etc.), componentsassociated with the magnetic separation device (e.g., motors configuredto position the magnetic field source and magnetic field guides), andother components as desired.

The systems may generally include one or more magnetic separationdevices as described herein and a processor configured to control theone or more magnetic separation devices. These two components may beintegrated into the same article of manufacture as a single unit, ordistributed among two or more different units (e.g., as a system) wherethe two or more different units are in communication with each other,e.g., via a wired or wireless communication protocol.

Accordingly, aspects of the present disclosure further include systems,e.g., computer based systems, which are configured to separatemagnetically labeled moieties in a sample as described above. A“computer-based system” refers to the hardware, software, and datastorage devices used to analyze the information of the presentinvention. The minimum hardware of embodiments of the computer-basedsystems includes a central processing unit (CPU) (e.g., a processor), aninput device, an output device, and data storage device. Any one of thecurrently available computer-based systems may be suitable for use inthe embodiments disclosed herein. The data storage device may includeany manufacture including a recording of the present information asdescribed above, or a memory access means that can access such amanufacture. For example, embodiments of the subject systems may includethe following components: (a) a communications module for facilitatinginformation transfer between the system and one or more users, e.g., viaa user computer or workstation; and (b) a processing module forperforming one or more tasks involved in the analysis of themagnetically labeled moieties.

In addition to the magnetic separation device, systems of the presentdisclosure may include a number of additional components, such as dataoutput devices, e.g., monitors, printers, and/or speakers, data inputdevices, e.g., interface ports, a keyboard, a mouse, etc., fluidhandling components, power sources, etc.

Utility

The subject devices, methods, systems and kits find use in a variety ofdifferent applications where it is desirable to separate magneticallylabeled moieties from non-magnetically labeled moieties in a sample. Forexample, the subject devices, methods, systems and kits find use indetecting the presence of a moiety of interest in a sample. The moietyof interest may be magnetically labeled and then separated fromnon-magnetically labeled moieties (e.g., by being retained in theconduit while non-magnetically labeled moieties flow through theconduit) by using the devices, methods, systems and kits describedherein. In other embodiments, the moiety of interest is not magneticallylabeled and other moieties that are not of interest in the sample aremagnetically labeled. In these embodiments, the non-magnetically labeledmoieties of interest are not retained by the device and flow through theconduit, where they may be collected and/or further analyzed. Themagnetically labeled moieties that are not of interest are retained inthe conduit and thus separated from the non-magnetically labeledmoieties of interest.

In certain embodiments, the subject devices, methods, systems and kitsfind use in detecting a binding interaction of interest. In certainembodiments, the binding interaction is a binding interaction, such as,but not limited to, nucleic acid hybridization, a protein-proteininteraction, a receptor-ligand interaction, an enzyme-substrateinteraction, a protein-nucleic acid interaction, and the like. In someinstances, the subject methods, systems and kits find use in drugdevelopment protocols where the detecting a molecular bindinginteraction may be desired. For example, drug development protocols mayuse the subject devices, methods, systems and kits to detect molecularthe binding interactions between antibodies and antigens, orhybridization interactions between nucleic acids, or bindinginteractions between proteins, or binding interactions between receptorsand ligands, or binding interactions between enzymes and substrates, orbinding interactions between proteins and nucleic acids, and the like.For instance, detecting binding interactions such as these mayfacilitate the development of antibody-based drugs.

The subject devices, methods, systems and kits also find use indetecting molecular binding interactions between binding pairs that areincluded in complex samples. In some instances, the complex samples maybe analyzed directly without separating the binding molecules ofinterest from the other proteins or molecules that are not of interestthat are in the sample. In certain cases, proteins or molecules that arenot of interest are not bound by the magnetic labels and are notretained in the conduit of the magnetic separation device. Thus, thesubject devices, methods, systems and kits find use in assay protocolswhere complex samples may be used and where the binding interactions ofinterest may be detected with no purification of the sample necessaryfor detection of the binding interactions of interest.

Kits

Also provided are kits for practicing one or more embodiments of theabove-described methods and/or for use with embodiments of the devicesand systems described above. The subject kits may vary, and may includevarious components and reagents. Reagents and components of interestinclude those mentioned herein with respect to magnetic separationdevices or components thereof, and include, but are not limited to,magnetic labels (e.g., magnetic nanoparticles), binding agents, buffers,fluid flow conduits (e.g., disposable fluid flow conduits), etc.

In some instances, the kits include at least reagents finding use in themethods (e.g., as described above); and a computer readable mediumhaving a computer program stored thereon, wherein the computer program,when loaded into a computer, operates the computer to perform a magneticseparation assay as described herein; and a physical substrate having anaddress from which to obtain the computer program.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., CD, DVD, Blu-Ray, computer readable memory device (e.g., harddrive or flash memory), etc., on which the information has beenrecorded. Yet another means that may be present is a website addresswhich may be used via the Internet to access the information at aremoved site. Any convenient means may be present in the kits.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments of the present disclosure, and are notintended to limit the scope of what the inventors regard as theirinvention nor are they intended to represent that the experiments beloware all or the only experiments performed. Efforts have been made toensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

EXPERIMENTAL

Experiments were performed using a device for separating magneticallylabeled moieties in a sample, according to embodiments of the presentdisclosure.

Materials and Methods

Magnetic Separation Device

The magnetic separation device included six permanent magnets (N45 rareearth Neodymium (NdFeB) bar magnet, 2 in.×0.5 in.×0.5 in., CMSMagnetics, Inc.) and six wedge-shaped magnetic field guides. Themagnetic field guides were made of stainless steel and had a 60 degreeapex angle. The apex edges of each magnetic field guides had linearprofiles. The six permanent magnets were arranged into two sets of threemagnets. Each set of three magnets had overall dimensions of 6 in.×0.5in.×0.5 in. The first and second sets of magnets were positioneddirectly opposite from each other in the device. Each permanent magnethad a corresponding magnetic field guide attached, and the apex edges ofthe first set of magnetic field guides were directly opposite andparallel to the apex edges of the second set of magnetic field guides.During the separation assay, the gap between the apex edges of the twosets of magnetic field guides was 1 mm. The magnetic flux density in thegap between the apex edges of the magnetic field guides was measured tobe 1.1 Tesla and the magnetic field gradient was 0.8 T/mm. The magneticflux was localized in the gap between the apex edges of the magneticfield guides with a direction going from the first set of magnets to thesecond set of magnets.

Conduit

The conduit was silicone tubing with a 3 mm outer diameter and a 2 mminner diameter. The effective length of the conduit was 6 inches, whichcorresponds to the length of the set of magnets in contact with theconduit.

Reagents and Sample for Separating Magnetically Labeled Moieties in aSample

BD Imag™ Human CD4 T Lymphocyte Enrichment Set (Becton, Dickinson andCo.) that included biotin human CD4 T lymphocyte enrichment assaymixture and streptavidin coated magnetic particles was used in theexperiments. The magnetic particles were superparamagnetic particleshaving an average diameter ranging from 200 nm to 400 nm and a stockconcentration of 200 μg/ml. According to the manufacturer's recommendedprotocol, 5 μl of the cocktail was used per million cells. Afterincubation and washing, 5 μl streptavidin coated magnetic particles wasused per million cells. The experimental sample was human peripheralblood mononuclear cells (PBMCs) prepared by using BD Vacutainer® CPT™Cell Preparation Tube with Sodium Citrate (Becton, Dickinson and Co.).The PBMCs were suspended in 1× phosphate buffered saline (PBS) with 0.5%bovine serum albumin (BSA) and 20 mM EDTA in a concentration of 2 to 50million cells per ml. Biotinylated antibodies in the assay mixture boundto all populations in PBMCs except CD4+T lymphocytes. Streptavidin thatwas conjugated to magnetic particles in the assay mixture bound tobiotin specifically. With the above two step binding method, all cellsin PBMCs except CD4+T lymphocytes were magnetically labeled by thespecific binding interaction between the biotin labeled PBMCs and thestreptavidin coated magnetic particles.

The sample was flowed through the conduit positioned in between themagnetic field guides in a magnetic separation device and magneticallylabeled cells were captured in the magnetic field in the gap between theapex edges of the magnetic field guides. The CD4+T lymphocytes, whichwere not magnetically labeled, were not retained in the conduit andpassed through the conduit to a detector or collection tube positioneddownstream from the magnets. When the separation was finished, theconduit was removed from between the magnetic field guides and thecaptured cells were flushed out with buffer under higher pressure.Because human T regulatory cells are a small subpopulation of CD4+Tlymphocytes, magnetic depletion of non CD4+T lymphocytes is a way toenrich T regulatory cells in a sample.

Operation Conditions

Magnetically labeled cells and non-labeled cells passed through themagnetic separation device in a conduit driven by a peristaltic pump orair pressure. The flow rate was 200 μl/min to 400 μl/min. For example,an air compressor was used to apply 18 psi to the sample in the conduitto achieve a 400 μl/min flow rate in the conduit. With 2 to 50 millionPBMCs in 1 ml sample volume, the separation and recovery was completedin 10 minutes or less. The experiment was performed at room temperature.Optionally, the labeled PBMCs were kept on ice before performing theseparation assay.

Results

Both magnetically captured cells and non-captured cells were analyzedwith a flow cytometer after fluorescent staining. The results indicatedthat the magnetic separation device had a 98% separation efficiency and90% of the T regulatory cells were recovered.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1-5. (canceled)
 6. A method of separating magnetically labeled moietiesin a sample, the method comprising: (a) positioning in a magneticseparation device a conduit configured to direct a flow of a samplethrough the magnetic separation device, the magnetic separation devicecomprising: (i) a magnetic field source; (ii) a first magnetic fieldguide having a wedge-shaped portion with an apex edge; and (iii) asecond magnetic field guide having a wedge-shaped portion with an apexedge, wherein one or more of the first and second magnetic field guidesis configured to increase a magnetic flux from the magnetic fieldsource, and the apex edge of the first magnetic field guide is proximalto and substantially parallel to the apex edge of the second magneticfield guide; and (b) applying a magnetic field to separate magneticallylabeled moieties from non-magnetically labeled moieties in the sample.7. The method of claim 6, further comprising: positioning the conduitaway from the magnetic field; and recovering the magnetically labeledmoieties retained in the conduit.
 8. The method of claim 7, wherein thepositioning the conduit away from the magnetic field comprises one ormore of removing the conduit from the device and moving the magneticfield source away from the conduit.
 9. The method of claim 6, furthercomprising specifically attaching a magnetic label to target moieties inthe sample prior to applying the magnetic field to the sample.
 10. Themethod of claim 6, wherein the sample comprises a biological sample. 11.A system for separating magnetically labeled moieties in a sample, thesystem comprising: (a) one or more magnetic separation devices forseparating magnetically labeled moieties in the sample, wherein each ofthe one or more magnetic separation devices comprises: (i) a magneticfield source; (ii) a first magnetic field guide having a wedge-shapedportion with an apex edge; and (iii) a second magnetic field guidehaving a wedge-shaped portion with an apex edge, wherein one or more ofthe first and second magnetic field guides is configured to increase amagnetic flux from the magnetic field source, the apex edge of the firstmagnetic field guide is proximal to and substantially parallel to theapex edge of the second magnetic field guide, and the device isconfigured to separate magnetically labeled moieties fromnon-magnetically labeled moieties in the sample; and (b) a conduitpositioned in the magnetic separation device and configured to direct aflow of the sample through the magnetic separation device.
 12. Thesystem of claim 11, wherein the conduit is positioned such that alongitudinal axis of the conduit is substantially parallel to alongitudinal axis of the first magnetic field guide and a longitudinalaxis of the second magnetic field guide.
 13. The system of claim 11,wherein the conduit is substantially free from magnetic gradientenhancing materials.
 14. The system of claim 11, wherein the systemcomprises a first magnetic separation device and a second magneticseparation device arranged downstream from the first magnetic separationdevice.
 15. The system of claim 11, further comprising a flow cytometerarranged downstream from the one or more magnetic separation devices.16. The method of claim 6, wherein the conduit comprises a single linearflow channel having a longitudinal axis that is parallel to alongitudinal axis of the first magnetic field guide and a longitudinalaxis of the second magnetic field guide.
 17. The method of claim 6,wherein the first and second magnetic field guides have cross-sectionalprofiles that taper to a point or are rounded at their apex edges. 18.The method of claim 6, wherein the apex edges of the first and secondmagnetic field guides each have a linear profile or a saw-tooth profile.19. The method of claim 6, wherein the first and second magnetic fieldguides each comprise a soft magnet, and the magnetic field sourcecomprises a permanent magnet.
 20. The method of claim 6, wherein themagnetic separation device further comprises a second magnetic fieldsource, wherein the first magnetic field guide is disposed on a surfaceof the magnetic field source facing the second magnetic field guide andis configured to increase the magnetic flux from the magnetic fieldsource, and the second magnetic field guide is disposed on a surface ofthe second magnetic field source facing the first magnetic field guideis configured to increase a magnetic flux from the second magnetic fieldsource.
 21. The method of claim 6, wherein the conduit is positionedbetween the first and second magnetic field guides such that its centrallongitudinal axis is equidistant from the apex edges of the first andsecond magnetic field guides.
 22. The method of claim 6, wherein theconduit has a rectangular cross-sectional profile.
 23. The method ofclaim 6, wherein the conduit has a cross-sectional area upstream fromthe portion of the conduit positioned between the magnetic field guidesthat is greater than the cross-sectional area of the portion of theconduit positioned between the magnetic field guides.
 24. The method ofclaim 6, wherein the conduit is operatively coupled to a conduit holder.25. The method of claim 6, wherein the first magnetic field source andthe second magnetic field source have magnetization vectors aligned insubstantially the same direction.